Charakterisierung des Transkriptionsfaktors FoxO als zentraler Stressmediator und Effektor einer stressinduzierten Immunantwort im respiratorischen Epithel der Taufliege Drosophila melanogaster

FoxO-Proteine gehören zur umfangreichen Familie der Forkhead Box-Transkriptions-faktoren. Den vier FoxO-Proteinen des Menschen, die als FoxO1a, FoxO3a, FoxO4 und FoxO6 bezeichnet werden, steht in der Taufliege Drosophila melanogaster ein einziges Homolog gegenüber. Bei den FoxO-Transkriptionsfaktoren handelt es sich um eine evolutionär sehr alte und hochkonservierte Gruppe von Proteinen. Ihnen gemein ist eine zentrale Rolle, die sie in der Regulation metabolischer Prozesse der Zelle einnehmen. Sie werden durch die aktivierte Insulin/IGF-Signalkaskade inhibiert und kontrollieren unterschiedliche zelluläre Eigenschaften. Insbesondere sind sie bei der Inhibition des Zellzyklus, der Antwort auf oxidativen Stress, bei der Apoptose und in verschiedenen Aspekten des Metabolismus von elementarer Bedeutung. In Drosophila wurde auch eine Beteiligung von FoxO an Reaktionen des angeborenen Immunsystems gezeigt. Im Rahmen der vorliegenden Arbeit konnte FoxO als zentraler Vermittler der zellulären Antwort auf Stress identifiziert werden, der unspezifisch durch eine heterogene Auswahl von Stressoren aktiviert wird. Dabei handelt es sich um einen evolutionär hochkonservierten Prozess, der in respiratorischen Epithelien der Taufliege und des Menschen gleichermaßen zu funktionieren scheint. FoxO konnte aber nicht nur als Schlüsselmolekül der Reaktion auf Umweltstressoren in Barriereepithelien identifiziert werden, sondern auch als Vermittler einer durch Stress induzierten Immunreaktion. Der zentrale Verteidigungsmechanismus gegen eindringende Mikroorganismen in Drosophila ist die Synthese Antimikrobieller Peptide (AMPs). Ihre Expression wird durch zwei intrazelluläre Signalkaskaden (den Toll- und den IMD-Signalweg) vermittelt durch NF-B-Transkriptionsfaktoren, reguliert. Eine Aktivierung dieser elementaren Immunreaktion kann aber auch in Abwesenheit einer Infektion und unabhängig von den NF-B-Faktoren, durch Stress unter der Beteiligung des Transkriptionsfaktors FoxO induziert werden. Zwei Stressoren, die eine derartige Immunantwort auslösen können, wurden in dieser Arbeit identifiziert; es handelt sich um metabolischen und oxidativen Stress. Diese beiden Stressoren rufen jedoch gegensätzliche Funktionen des FoxO-Proteins hervor. Während metabolischer Stress in Form von Nahrungsentzug eine direkte FoxO-induzierte AMP-Expression initiiert, erfährt die durch oxidativen Stress hervorgerufene AMP-Expression eine negative Regulation durch FoxO. Die Entdeckung eines bislang unbekannten, NF-B-Signalweg-unabhängigen Regulators des angeborenen Immunsystems, eröffnet neue Perspektiven der Immunregulation und präsentiert mit FoxO ein Bindeglied zwischen zellulärem Stress und dem angeborenen Immunsystem. Damit wurde ein Mechanismus entdeckt, der eine Anpassung der Immunabwehr an wechselnde Umweltbedingungen ermöglicht.FoxO proteins are members of a large family of forkhead box transcription factors. The four human FoxOs, referred to as FoxO1a, FoxO3a, FoxO4 and FoxO6, are faced by a single homolog in the fruit fly Drosophila melanogaster, and represent a group of evolutionary ancient and highly conserved proteins. A central function of FoxO transcription factors is the regulation of metabolic processes within the cell, thus, they are inhibited by Insulin/IGF pathway activity. In addition, these factors control various cellular mechanisms, such as cellcycle inhibition, oxidative stress response, and apoptosis. In Drosophila FoxO seems also to be involved in innate immune reactions. This work identified FoxO as a central mediator of cellular responses to stress, which is generally activated by heterogeneous stress conditions. This newly-characterized function is an evolutionary highly conserved process, which seems to operate in respiratory epithelial cells of Drosophila and human likewise. However, FoxO was not only identified as a key molecule in the response to environmental stressors in barrier epithelia, but also as a mediator of a stress-induced immune reaction. In Drosophila, the main defense reaction against invading microorganisms is the synthesis of antimicrobial peptides (AMPs). Usually, the AMP gene expression is regulated by two intracellular signaling pathways - the Toll- and the IMD-pathway, mediated by NF-B transcription factors. Here, I demonstrate that an activation of this basic immune reaction can also be induced in an infection- and NF-B-independent manner, namely by stress with the participation of FoxO. As part of this work two stress factors, metabolic and oxidative stress, were identified that are able to induce an immune response. Interestingly, FoxO is involved differentially in the reaction to these two stress conditions. Whereas metabolic stress, induced by starvation, results in an AMP expression dependent on FoxO, this transcription factor negatively regulates oxidative stress-induced AMP expression. The characterization of an innovative innate immune regulation process by FoxO, which works independently of the canonical NF-B signaling cascades, offers new perspectives in immune regulation and presents a connection between cellular stress and innate immunity. This mechanism allows a direct adjustment of the immune reaction to alternating environmental conditions

Dermatophagoides pteronyssinus Major Allergen 1 Activates the Innate Immune Response of the Fruit Fly Drosophila melanogaster

Some allergens with relevant protease activity have the potential to directly interact with host structures. It remains to be elucidated whether this activity is relevant for developing their allergenic properties. The major goal of this study was to elucidate whether allergens with a strong protease activity directly interact with modules of the innate immune system, thereby inducing an immune response. We chose Drosophila melanogaster for our experiments to prevent the results from being influenced by the adaptive immune system and used the armamentarium of methods available for the fly to study the underlying mechanisms. We show that Dermatophagoides pteronyssinus major allergen 1 (Der p 1), the major allergen of the house dust mite, efficiently activates various facets of the Drosophila innate-immune system, including both epithelial and systemic responses. These responses depend on the immune deficiency (IMD) pathway via activation of the NF-κB transcription factor Relish. In addition, the major pathogen associated molecular pattern recognizing receptor of the IMD pathway, peptidoglycan recognition protein-LC, was necessary for this response. We showed that Der p 1, which has cysteine protease activity, cleaves the ectodomain of peptidoglycan recognition protein-LC and, thus, activates the IMD pathway to induce a profound immune response. We conclude that the innate immune response to this allergen-mediated proteolytic cleavage represents an ancient type of danger signaling that may be highly relevant for the primary allergenicity of compounds such as Der p 1

Adult and Larval Tracheal Systems Exhibit Different Molecular Architectures in Drosophila

Knowing the molecular makeup of an organ system is required for its in-depth understanding. We analyzed the molecular repertoire of the adult tracheal system of the fruit fly Drosophila melanogaster using transcriptome studies to advance our knowledge of the adult insect tracheal system. Comparing this to the larval tracheal system revealed several major differences that likely influence organ function. During the transition from larval to adult tracheal system, a shift in the expression of genes responsible for the formation of cuticular structure occurs. This change in transcript composition manifests in the physical properties of cuticular structures of the adult trachea. Enhanced tonic activation of the immune system is observed in the adult trachea, which encompasses the increased expression of antimicrobial peptides. In addition, modulatory processes are conspicuous, in this case mainly by the increased expression of G protein-coupled receptors in the adult trachea. Finally, all components of a peripheral circadian clock are present in the adult tracheal system, which is not the case in the larval tracheal system. Comparative analysis of driver lines targeting the adult tracheal system revealed that even the canonical tracheal driver line breathless (btl)-Gal4 is not able to target all parts of the adult tracheal system. Here, we have uncovered a specific transcriptome pattern of the adult tracheal system and provide this dataset as a basis for further analyses of the adult insect tracheal system

Temporal Expression and Localization Patterns of Variant Surface Antigens in Clinical <em>Plasmodium falciparum</em> Isolates during Erythrocyte Schizogony

<div><p>Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite <em>Plasmodium falciparum</em> possesses a number of multi-copy gene families, including <em>var</em>, <em>rif</em>, <em>stevor</em> and <em>pfmc-2tm,</em> which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on <em>in vitro</em> analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by <em>P. falciparum</em> in the human host. To investigate the expression of the <em>var</em>, <em>rif-A</em>, <em>rif-B</em>, <em>stevor</em> and <em>pfmc-2tm</em> family genes under conditions that mimic more closely the natural course of infection, <em>ex vivo</em> clinical <em>P. falciparum</em> isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle <em>in vitro</em> were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families <em>rif-A</em>, <em>stevor</em> and <em>pfmc-2tm</em> at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the <em>pfmc-2tm</em> family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and <em>Pf</em>MC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of <em>P. falciparum</em> multi-copy gene families during clinical progression and are suggestive of diverse functional roles of the respective proteins.</p> </div

Immunoblot analysis of VSA abundance in the clinical isolate #5 and the 3D7 strain.

<p><b>A, B</b>: Isolate #5 (h: time of <i>in vitro</i> cultivation) and 3D7 (hpi: hours post infection) at successive developmental stages were harvested (<b>A</b>), and differences in VSA abundance in the membrane fraction were assessed by immunoblot using α-RIF40, α-RIF44, α-RIF50, α-STEVOR-mix, α-<i>Pf</i>MC-2TM-SC, and α-<i>Pf</i>MC-2TM-CT antisera (<b>B</b>). As expected, RIFIN and STEVOR were present at higher levels in the clinical isolate; in contrast, <i>Pf</i>MC-2TM proteins were quantitatively increased in 3D7 parasites. Differences were most obvious in pigmented parasite stages (trophozoites, schizonts, left), which also exhibited the highest levels of protein during intraerythrocyic development, but upon longer exposure (*), similar results were also observed for ring stage parasites (right). The luminal endoplasmic reticulum (ER) protein BiP (HSP70) served as a loading control.</p

Proportion of infected erythrocytes with VSAs exported to host cell compartments in clinical isolates and 3D7 at different developmental stages.

1<p>Time point not determined for isolate #1.</p>2<p>Time point of 44 h for isolate #4.</p><p>hpi, hours post-infection.</p

Expression of multi-copy gene families in the 3D7 laboratory strain during erythrocyte schizogony.

<p>Expression profiles of the multi-copy gene families <i>var</i> (red line, squares), <i>rif-A</i> (dark blue line, triangles), <i>rif-B</i> (light blue, upside-down triangles), <i>stevor</i> (green line, diamonds), and <i>pfmc-2tm</i> (yellow line, dots) in long-term <i>in vitro</i> cultivated strain 3D7 during erythrocyte schizogony. In contrast to the clinical isolates, 3D7 parasites exhibited a slight increase of <i>rif-A</i>, <i>stevor</i> and <i>pfmc-2tm</i> expression during the ring stage, and the main peak of expression of the multi-copy gene families was observed in mid-age trophozoites. Transcription of the <i>var</i> gene family was restricted to ring stage parasites. Expression was normalized to the reference gene <i>fructose-bisphosphate aldolase</i> and is represented by either ΔCt (left chart) or relative expression (RELATEXP, right chart). 3D7 time course experiments were performed twice and two samples were analysed for each time point at least in duplicates. Graphically shown are mean values and standard deviations of all qPCR runs performed for the respective time points Parasite developmental age (hpi) and the corresponding parasitic stage (shown as Giemsa staining) are plotted on the x-axis.</p

Validation of the degenerate primer pairs used in quantitative real-time PCR.

<p>Primer pairs targeting the multi-copy gene families <i>var</i>, <i>rif-A</i>, <i>rif-B</i>, <i>stevor</i> and <i>pfmc-2tm</i> were designed to amplify a broad repertoire of different genes present in the 3D7 genome (first bar, 1), as indicated by <i>in silico</i> PCR results (second bar, 2). Experimental validation revealed similar numbers of genes amplified by the indicated primer pairs in every <i>P. falciparum</i> genotype relative to single-copy <i>fructose-bisphosphate aldolase</i> (RELATNO) (third to seventh bar, 3–7). <i>In silico</i> PCR results were experimentally confirmed for the <i>var</i> (red), <i>stevor</i> (green) and <i>pfmc-2tm</i> (yellow) primer pairs; however, the <i>rif-A</i> (dark blue) and <i>rif-B</i> (light blue) primer pairs only partially covered the genomic repertoire. Shown are mean values and standard deviations obtained by analysing two biological samples of 3D7 and <i>ex vivo</i> isolated gDNA of the clinical isolates in quadruplicates. 1: Number of genes present in the 3D7 genome (set as 100%); 2: number and percentage of genes amplified <i>in silico</i> using the one mismatch configuration (<a href="http://insilico.ehu.es" target="_blank">http://insilico.ehu.es</a>); 3–7: experimentally calculated RELATNOs of amplified genes of the indicated multi-copy gene families using gDNA from the 3D7 laboratory strain (3) (including percentages) and from clinical isolates #1 (4), #2 (5), #3 (6) and #4 (7).</p

Localization of VSAs during the intraerythrocytic developmental cycle.

<p><b>A-C:</b> Representative immunofluorescence images of the indicated VSAs in different parasite developmental stages of clinical isolate #4 (<b>A</b>), 3D7 parasites (<b>B</b>), and free merozoites from isolate #1 (<b>C</b>). First row: Giemsa staining of the corresponding parasitic stage. Second row: Positive control serum obtained from a semi-immune patient. Third row: <i>Pf</i>EMP1-specific antibody, showing the presence of the protein in Maurer’s clefts over the entire time course (<b>A, B</b>). Third to eighth rows: 2TM proteins were exported into the host cell (12–36 hpi) during the trophozoite stage but remained inside the parasite in the schizont stage (48 hpi). Proteins of the RIFIN-A family frequently localized to Maurer’s clefts, particularly when using the α-RIF29n antiserum, and the erythrocyte membrane; STEVOR and <i>Pf</i>MC-2TM localized predominantly to the erythrocyte membrane (<b>A, B</b>). RIFIN and STEVOR proteins were also observed at the apical tip or at the merozoite membrane, respectively. Isolate #1 also exhibited <i>Pf</i>MC-2TM-specific fluorescence in free merozoites when using the α-P<i>f</i>MC-2TM-CT antiserum (<b>C</b>). All antibodies were visualized with Alexa488-conjugated secondary antibody (green), and nuclei were stained with DAPI (blue).</p